TUMOR-NECROSIS-FACTOR INCREASES HEPATOCELLULAR GLUTATHIONE BY TRANSCRIPTIONAL REGULATION OF THE HEAVY SUBUNIT CHAIN OF GAMMA-GLUTAMYLCYSTEINE SYNTHETASE

Tumor necrosis factor (TNF) is an inflammatory cytokine that causes ce ll injury by generation of oxidative stress, Since glutathione (GSH) i s a key cellular antioxidant that detoxifies reactive oxygen species, the purpose of our work was to examine the regulation of cellular GSH, the expression of heavy subunit chain of gamma-glutamylcysteine synth etase (gamma-GCS-HS), and control of intracellular generation of react ive oxygen species in cultured rat hepatocytes treated with TNF. Expos ure of cells to TNF (10,000 units/ml) resulted in depletion of cellula r GSH levels (50-70%) and overproduction of hydrogen peroxide (2-3-fol d) and lipid peroxidation, However, cells treated with lower doses of TNF (250-500 units/ml) exhibited increased levels of GSH (60-80% over control). TNF treatment increased (70-100%) the levels of gamma-GCS-HS mRNA, the catalytic subunit of the regulating enzyme in GSH biosynthe sis. Furthermore, intact nuclei isolated from hepatocytes treated with TNF transcribed the gamma-GCS-HS gene to a greater extent than contro l cells, indicating that TNF regulates gamma-GCS-HS at the transcripti onal level. The capacity to synthesize GSH de novo determined in cell- free extracts incubated with GSH precursors was greater (50-70%) in he patocytes that were treated with TNF; however, the activity of GSH syn thetase remained unaltered by TNF treatment indicating that TNF select ively increased the activity of gamma-GCS. Despite activation of nucle ar factor-kappa B (NF-kappa B) by TNF, this transcription factor was n ot required for TNF-induced transcription of gamma-GCS-HS as revealed by deletion constructs of the gamma-GCS-HS promoter subcloned in a chl oramphenicol acetyltransferase reporter vector and transfected into He pG2 cells, In contrast, a construct containing AP-1 like/metal respons e regulatory elements increased chloramphenicol acetyltransferase acti vity upon exposure to TNF. Thus, TNF increases hepatocellular GSH leve ls by transcriptional regulation of gamma-GCS-HS gene, probably throug h AP-1/metal response element-like binding site(s) in its promoter, wh ich may constitute a protective mechanism in the control of oxidative stress induced by inflammatory cytokines.